1. Field of the Invention
The present invention in the fields of molecular biology, immunology and medicine relates to combinations or mixtures of nucleic acid molecules and chimeric nucleic acid molecules that encode an antigen and a small interfering RNA (siRNA). The expression of the siRNA blocks expression of one or more an anti-apoptotic protein in vivo. This results in prolonging the life of important antigen presenting cells, dendritic cells (DCs), and as a consequence, the more potent induction and enhancement immune responses, primarily cytotoxic T lymphocyte (CTL) responses to specific antigens such as tumor or viral antigens.
2. Description of the Background Art
Cytotoxic T lymphocytes (CTL) are critical effectors of anti-viral and antitumor responses (reviewed in Chen, C H et al., J Biomed Sci. 5: 231-252, 1998; Pardoll, D M. Nat Med. 4: 525-531, 1998; Wang, R F et al., Immunol Rev. 170: 85-100, 1999). Activated CTL are effector cells that mediate antitumor immunity by direct lysis of their target tumor cells or virus-infected cells and by releasing of cytokines that orchestrate immune and inflammatory responses that interfere with tumor growth or metastasis, or viral spread. Depletion of CD8+ CTL leads to the loss of antitumor effects of several cancer vaccines (Lin, K-Y et al., Canc Res 56: 21-26, 1996; Chen, C-H et al., Canc Res. 60: 1035-42, 2000). Therefore, the enhancement of antigen presentation through the MHC class I pathway to CD8+ T cells has been a primary focus of cancer immunotherapy.
Naked DNA vaccines have emerged recently as attractive approaches for vaccine development (reviewed in Hoffman, S L et al., Ann NY Acad Sci 772: 88-94, 1995; Robinson, H L. Vaccine 15: 785-787, 1997; Donnelly, J J et al., Annu Rev Immunol 15: 617-648, 1997; Klinman, D M et al., Immunity 11: 123-129, 1999; Restifo, N P et al., Gene Ther 7: 89-92, 2000; Gurunathan, S et al., Annu Rev Immunol 18: 927-974, 2000). DNA vaccines generated long-term cell-mediated immunity (reviewed in Gurunathan, S et al., Curr Opin Immunol 12: 442-447, 2000) and can generate CD8+ T cell responses in vaccinated humans (Wang, R et al. Science 282: 476-480, 1998).
However, one limitation of these vaccines is their lack of potency, since the DNA vaccine vectors generally do not have the intrinsic ability to be amplified and to spread in vivo as do some replicating viral vaccine vectors. Furthermore, some tumor antigens such as the E7 and E6 proteins of human papillomavirus-16 (“HPV-16”) are weak immunogens (Chen et al., 2000, supra). Therefore, there is a need in the art for strategies to enhance DNA vaccine potency, particularly for more effective cancer and viral immunotherapy.
The present inventors and their colleagues demonstrated that linkage of HPV-16 E7 antigen to a number of immunogenicity-potentiating polypeptides (Kim J W et al., Gene Ther. 11:1011-18, 2004,), such as Mycobacterium tuberculosis (Mtb) heat shock protein 70 (Hsp70) (Chen et al., supra; Wu et al., WO 01/29233) and CRT (Cheng W F et al., J Clin Invest, 2001, 108:669-78; WO/0212281) result in the enhancement of DNA vaccine potency. See, also Cheng W F et al., Vaccine 23:3864-74, 2005; Peng S et al., J Biomed Sci. 12:689-700, 2005; Peng S et al., J Virol. 2004, 78:8468-76; Peng S et al., Gene Ther. 2005 (Sep. 22; Epublished ahead of print)
Others have shown, using protein vaccines, as distinct from DNA immunogens, that immunization with HSP complexes isolated from tumor or virus-infected cells potentiated anti-tumor immunity (Janetzki, S et al., J Immunother 21:269-7, 1998) or antiviral immunity (Heikema, A et al., Immunol Lett 57:69-74, 1997). Immunogenic HSP-peptide complexes could be reconstituted in vitro by mixing the peptides with HSPs (Ciupitu, A M et al., 1998. J Exp Med 187:685-9, 1998). HSP-based protein vaccines have been created by fusing antigens to HSPs (Suzue, K et al., J Immunol 156:873-79, 1996). However, prior to the discoveries of the present inventors and their colleagues since about 1999 with DNA immunogens, HSP vaccines (and those employing other intracellular transport proteins or intercellular spreading proteins) were limited to peptide/protein molecules that were typically produced bacteria using bacterial expression vectors and purified therefrom. The present inventors and their colleagues were the first to provide naked DNA and self-replicating RNA vaccines that incorporated HSP70 and other immunogenicity-potentiating polypeptides. The present inventors and their colleagues were also the first to demonstrate that linking antigen to intracellular targeting moieties calreticulin (CRT), domain II of Pseudomonas aeruginosa exotoxin A (ETA(dII)), or the sorting signal of the lysosome-associated membrane protein type 1 (Sig/LAMP-1) enhanced DNA vaccine potency compared to compositions comprising only DNA encoding the antigen of interest. To enhance MHC class II antigen processing, one of the present inventors and colleagues (Lin, K Y et al., 1996, Canc Res 56: 21-26) linked the sorting signals of the lysosome-associated membrane protein (LAMP-1) to the cytoplasmic/nuclear human papilloma virus (HPV-16) E7 antigen, creating a chimera (Sig/E7/LAMP-1). Expression of this chimera in vitro and in vivo with a recombinant vaccinia vector had targeted E7 to endosomal and lysosomal compartments and enhanced MHC class II presentation to CD4+ T cells. This vector was found to induce in vivo protection against an E7+ tumor, TC-1 so that 80% of mice vaccinated with the chimeric Sig/E7/LAMP1 vaccinia remained tumor free 3 months after tumor injection. Treatment with the Sig/E7/LAMP-1 vaccinia vaccine cured mice with small established TC-1 tumors, whereas the wild-type E7-vaccinia showed no effect on this established tumor burden. These findings point to the importance of adding an immunopotentiating “element” (in the form of DNA encoding that “element”) to DNA encoding an antigen to enhance in vivo potency of a recombinant DNA vaccine for antigens that are presented as either MHC class I- or MHC class II-antigen complexes, such as by rerouting a cytosolic tumor antigen to the endosomal/lysosomal compartment.
Intradermal administration of DNA vaccines via gene gun can efficiently deliver genes of interest into professional antigen presenting cells (APCs) in vivo (Condon C et al., Nat Med, 2: 1122-28, 1996). The skin contains numerous bone marrow-derived APCs (called Langerhans cells) that are able to move through the lymphatic system from the site of injection to draining lymph nodes (LNs), where they can prime antigen-specific T cells (Porgador A et al., J Exp Med 188: 1075-1082, 1998). Powerful APCs in other sites, particularly in lymphatic tissue are dendritic cells (DC). Gene gun immunization therefore provides the opportunity to test vaccine strategies that require direct delivery of DNA or RNA to APCs.
Antigen presentation by DCs is a critical element for the induction of the cellular immune responses that mediate various types of immunotherapy, particularly tumor immunotherapy. Several studies demonstrated that immunization with tumor antigen-pulsed DCs could break the tolerance of the immune system against antigens expressed by tumor cells and in some cases generate appreciable clinical responses. Thus, DC-based vaccines represent a promising method for the treatment of malignancies. See, for example, Gunzer, M et al., Crit Rev Immunol 21: 133-45, 2001; Engleman, E G Dendritic cell-based cancer immunotherapy. Semin Oncol 30:23-29, 2003; Schuler, G et al., Curr Opin Immunol 15:138-147, 2003; Cerundolo, V et al., Dendritic cells: a journey from laboratory to clinic. Nat Immunol 5:7-10, 2004; Figdor, C G et al., Nat Med 10:475-480, 2004; Markiewicz, M A et al., Cancer Invest 22:417-434, 2004; Turtle, C J et al., Curr Drug Targets 5:17-39, 2004).
Dendritic cell-based vaccines have become an important approach for the treatment of malignancies. Numerous techniques have recently been designed to optimize dendritic cell activation, tumor antigen delivery to dendritic cells, and induction of tumor-specific immune responses in vivo. Dendritic cells, however, have a limited life span because they are subject to apoptotic cell death mediated by T cells, hindering their long-term ability to prime antigen-specific T cells.
DCs, however, have a limited life span that hinders their long-term ability to prime antigen-specific T cells (see Ronchese, F et al. J Exp Med 194:F23-26, 2001). A principal contributor to the shortened lifespan of DCs is CTL-induced apoptosis. After activation by DCs, CTLs that recognize epitopes can kill target cells expressing these epitopes, typically presented by MHC Class I proteins. Because DCs express MHC-I:antigen peptide complexes, newly primed CTLs can kill the very DCs that activated them (Medema, J P et al., J Exp Med 194:657-667, 2001). Thus, DC-based vaccination should be enhanced by inhibiting apoptosis and prolong survival of antigen-expressing DCs in vivo (Kim, T W et al., J Immunol 171:2970-2976, 2003a; Kim, T W et al., J Clin Invest 112:109-17, 2003(b); and a patent application by the present inventors and colleagues WO05/047501 (26 May 2005) incorporated herein by reference in its entirety.
The present inventors and their colleagues have used gene gun immunization of DNA compositions to test vaccine strategies that involve intracellular targeting strategies that direct delivery of DNA or RNA to APCs. The targeting molecules (using coding DNA linked to DNA encoding an antigen) that have shown potent effects include Mycobacterium tuberculosis heat shock protein 70 (HSP70) (Chen C H et al., 2000, Cancer Res 60:1035-42, 2000), calreticulin (CRT; Cheng W F, 2001, supra), and the sorting signal of the lysosome-associated membrane protein 1 (LAMP-1; Ji H et al., Hum Gene Therapy, 10:2727-40, 1999).
Vaccination with DNA vectors that encode such fusion proteins are able to route an antigen (generally exemplified with HPV-16 E6 and E7) to desired subcellular compartments, and enhance antigen processing and presentation to T cells. Therefore, direct delivery of DNA vaccines into DCs via gene gun provides an opportunity to modify the quality and quantity of DNA-transfected DCs and influence vaccine potency.
T cell-mediated apoptotic cell death can occur through two major pathways, the intrinsic and the extrinsic pathways. See, for example, Russell, J H et al., Annu Rev Immunol 20:323-370, 2002). In general, death domain-containing receptors such as CD95 (APO-1/Fas) can sense the external signal (such as Fas ligand) and activate the extrinsic apoptotic pathway through the Fas-associated death domain (Fadd). This pathway is mediated by recruitment and activation of caspase-8, an initiator caspase, in the death-inducing signaling complex (DISC) followed by direct cleavage of downstream effector caspases.
The intrinsic pathway (granzyme B/perforin-mediated apoptosis), important for T cell-mediated induction of apoptotic DC death, initiates from within the cell. The pore-forming protein perforin and the serine protease granzyme B secreted into cells by antigen-specific CD8+ T cells induce intracellular changes, such as DNA damage, resulting in the release of a number of pro-apoptotic factors from mitochondria, such as cytochrome c, leading to the activation of another initiator caspase, caspase-9 (Jacotot, E et al., Ann NY Acad Sci 887:18-30, 1999; Korsmeyer, S J et al., Cell Death Differ 7:1166-73, 2000; Degli Esposti, M et al., Dive, C. Biochem Biophys Res Commun 304:455-61, 2003; Opferman J T et al., Nat Immunol 4: 410-15, 2003). 5-61, 2003; Opferman J T et al., Nat Immunol 4: 410-15, 2003). Activated caspase-9 leads to the activation of effector caspases (caspase-3, -6, and -7) in a protein complex called the apoptosome (for review, see Johnson, C R et al., Apoptosis 9:423-27, 2004) leading to proteolysis of a cascade of substrates and apoptotic death.
Thus Bak, Bax, and caspase 9 are clearly important pro-apoptotic proteins for the intrinsic apoptotic pathway and caspases-8 and -3 are is an important pro-apoptotic proteins in the extrinsic apoptotic pathway. Because of the role of Bak and Bax as gatekeepers in the intrinsic apoptotic pathway, the present inventors have conceived of targeting these genes for inhibition by RNA interference (RNAi) to diminish DC apoptosis. This is disclosed in detail and exemplified below. However, the present inventors conception includes a similar targeting of caspase-9, caspase-3 and caspase-8.
RNA interference (RNAi) is a recently reported phenomenon that has developed into a new approach for elucidating and regulating gene function. RNAi is a sequence-specific, post-transcriptional, gene-silencing mechanism that is effected through double-stranded RNA (dsRNA) molecules homologous to a sequence of the target gene (Elbashir, S M et al., Nature 411:494-498, 2001; Fire, A et al., Nature 391:806-811, 1998; Tuschl, T et al., Genes Dev 13:3191-3197, 1999). Fragments of the dsRNA called “small interfering” RNAs (siRNAs) can rapidly induce loss of function, and only a few molecules are required in a cell to produce the effect (Fire et al., supra) through hybrid formation between a homologous siRNA and mRNA (Lin, S L et al., Curr Cancer Drug Targets 1:241-247, 2001). A member of the RNase III family of nucleases named dicer has been identified as being involved in processing (Bernstein, E et al., Nature 409:363-366, 2001). DNA vector-mediated RNAi technology has made it possible to develop therapeutic applications for use in mammalian cells (Sui, G et al., Proc Natl Acad Sci USA 99:5515-5520, 2002; McCaffrey, A P et al., Nature 418:38-39, 2002; Lee, N S et al., Nat Biotechnol 20:500-505, 2002). There have been several reports of delivery of siRNA by retroviral vectors for stable expression (Barton, G. M et al., Proc Natl Acad Sci USA 99:14943-14945, 2002; Paddison, P J et al., Cancer Cell 2:17-23, 2002; Rubinson, D A et al., Nat Genet 33:401-406, 2003; Tiscornia, G et al., Proc Natl Acad Sci USA 100:1844-1848, 2003) or by adenoviral vectors for transient expression (Xia, H et al., Nat Biotechnol 20:1006-1010, 2002). RNAi may be effected by small interfering RNA molecules (siRNA) that induce sequence-specific degradation of mRNA or by inhibiting translation of its complementary mRNA (see, for example, Mittal V. Nat Rev Genetics 5:355-65, 2004). Use of this approach to prolong the life of DCs by targeting pro-apoptotic proteins with the appropriate siRNAs is one of the objects of the present invention.